This invention relates to detecting bacteria belonging to the genus Listeria. (The term "Listeria," as used herein, refers to the bacteria classified as such in Bergey's Manual of Systematic Bacteriology, with the exception of L. denitrificans, which recently has been removed from the genus Listeria based on molecular and phenotypic consideration (Rocourt et al. (1987) International Journal of Systematic Bacteriology (submitted)). Detection of Listeria bacteria is important in various medical and public health contexts. Listeria infection can cause a variety of symptoms ranging from cold like to flu-like, but is especially dangerous for a fetus, where it induces a 50% mortality rate.
According to a standard laboratory method and a method recommended by the F.D.A., the presence of Listeria in environmental or dairy specimens (e.g., milk) is detected by culturing an appropriately prepared sample on microbiological media under conditions favorable for growth of these organisms. The resulting colonies are examined for morphological and biochemical characteristics, a process that typically is started 48 hours after acquisition of the sample and takes between 9-19 days to complete.
Kohne et al. (1968) Biophysical Journal 8:1104-1118 discuss one method for preparing probes to rRNA sequences.
Pace and Campbell (1971) Journal of Bacteriology 107:543-547 discuss the homology of ribosomal ribonucleic acids from diverse bacterial species and a hybridization method for quantitating these homology levels.
Sogin, Sogin, and Woese (1972) Journal of Molecular Evolution 1:173-184) discuss the theoretical and practical aspects of using primary structural characterization of different ribosomal RNA molecules for evaluating phylogenetic relationships.
Fox, Pechman, and Woese (1977) International Journal of Systematic Bacteriology 27:44-57 discuss the comparative cataloging (of 16S ribosomal RNAs) approach to prokaryotic systematics.
The present invention will be better understood in light of the following definitions:
DNA--deoxyribonucleic acid, the type of nucleic acid containing deoxyribose as the sugar component.
RNA--ribonucleic acid, the type of nucleic acid containing ribose as the sugar component and which, most generally, is transcribed (copied) from DNA. RNA molecules may serve informational (e.g., messenger RNA), catalytic, or structural (e.g., ribosomal RNA, see below) cellular functions.
rRNA--Ribosomal RNA (rRNA) molecules are key elements of ribosomes, complex protein and RNA-containing "organelles" which, together with transfer RNAs, comprise the translation apparatus. Ribosomes are of profound importance to all organisms because they serve as the only known means of translating genetic information into cellular proteins, the main structural and catalytic elements of life.
Ribosomes contain three distinct RNA molecules which, in E. coli, are referred to as 5S, 16S and 23S rRNAs. These names historically are related to the size of the RNA molecules, as determined by sedimentation rate. However, they actually vary substantially in size between organisms. 5S, 16S, and 23S rRNA are commonly used as generic names for the homologous RNA molecules in any bacteria, and will be so used here.
Hybridization--the process by which, under defined reaction conditions, two partially or completely complementary nucleic acids are allowed to come together in an antiparallel fashion and form specific and stable hydrogen bonds. The stringency of a particular set of hybridization conditions is defined by the base composition of the probe/target duplex, as well as by the level and geometry of mispairing between the two nucleic acids. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and/or the temperature of hybridization.
Probe(s)--synthetic or biologically produced nucleic acids (DNA or RNA) which, by design or selection, contain specific nucleotide sequences that allow them to hybridize, under defined stringencies, specifically (i.e., preferentially) to target nucleic acid sequences.
Target--a nucleic acid sequence to which a particular probe is capable of preferentially hybridizing.
Other definitions are given as their first use arises in the text.